Study On Toxicity Mechanism Of Tilapia Liver DNA Damage And Apoptosis Of Zebrafish Eye By Cyantraniliprole

Cyantraniliprole is the second generation of ryanodine receptor inhibitor insecticides.It has broader and more efficient advantages for crops,which can effectively control lepidoptera,hemipteron and Coleoptera pests.Cyantraniliprole has endoscopic,osmotic,and conductive effects,which can be distributed throughout the plant and be efficient,fast-acting and long-lasting.As the global coverage rate of cyantraniliprole was increased,its use rate and risk to the environment were increases.So far,the toxicity effect of cyantraniliprole deficiency on the tilapia was unclear.The effect of cyantraniliprole on zebrafish has been deeply studied.Our laboratory has determined that cyantraniliprole will cause apoptosis of zebrafish eye cells.This paper evaluates the toxicity of cyantranclilprole on the tilapia and its mechanism of apoptosis of zebrafish eye cells,using the tilapia and zebrafish embryos as test materials.The results of the experiment are as follows:1.The effect of cyantraniliprole on the tilapia(1)Acute toxicity of cyantraniliprole(95%)to tilapia 96h-LC50 was 37.82 mg·L-1 and were low toxicity.In chronic toxicity tests,cyantraniliprole inhibits the growth and development of tilapia,and over time the inhibition becomes more apparent.At 28 days,the blank control,solvent control,0.037mg·L-1,0.37 mg·L-1 and 3.7 mg·L-1 treated groups were 1.137%/d,0.948%/d,0.932%/d,0.819%/d,0.698%/d,respectively.The growth rate of the blank control group was faster than other groups,3.7mg·L-1 treatment group was slower than other groups and there had significant difference(p<0.01);(2)In chronic tests,the DNA damage of cyantraniliprole to liver cells of tilapia was detected by DAPI staining and single cell gel electrophoresis(SCGE).The results show that as the concentration of cyantraniliprole increases,the micronucleus rate increases significantly(p<0.01),and that the tail length,tail distance and tail DNA content of the comet increase significantly(p<0.01),which indicates that cyantraniliprole induce DNA damage to liver cells of tilapia,and with a good dose-effect relationship;(3)The RT-qPCR technique was used to study the effect of cyantraniliprole on DNA damage related genes to liver cells of tilapia larvae.The results show that cyantraniliprole can activate DNA damage and repair pathway,which causing DNA damage to liver cells and activating cell repair to DNA.The most up regulated genes were Ccna 1 and Rpa 3 in all tested 7 genes;(4)The results of the activity of antioxidant enzymes in liver cells of tilapia indicated that the content of reactive oxygen species(ROS)and malondialdehyde(MDA),and the activity of superoxide dismutase(SOD)、catalase(CAT)、peroxidase(POD)were increased.The results proposed that the effect of cyantraniliprole activated the oxidative stress response of liver cells and increased the activity of intracellular antioxidant enzymes;2.The study on the mechanism of apoptosis of zebrafish eye cells induced by cyantraniliprole(1)There are significantly different and relevant genes in the apoptosis pathway of zebrafish eye cells:pvalb1,rbl2,vax2,bmp4,pitx1,Imx1a,cyplbl,and b2m,where pvalbl and rbl2 are the most noticeable by the transcriptome sequencing.The gene pvalb1,rbl2,vax2,bmp4,pilx1,Imxla,cyplbl and b2m related to apoptosis of zebrafish eye cells were studied by RT-qPCR technology.The results show that the mRNA expression of these apoptosis-related genes was up regulated,gene rbl2 was the most one.These results proposed that cyantraniliprole affects the gene expression of the apoptosis pathway of zebrafish eye cells;(2)The protein expression level of 24 hpf zebrafish embryo rbl2 was tested after exposure by enzyme-linked reaction kit.The rbl2 content in the highest concentration treatment group(2.00 mg·L-1)was 7.41 pg·mL-1 and has significantly different from it of the control group(3.54 pg·mL-1)(p<0.01).The results proposed that cyantraniliprole affect the rbl2 protein expression level;(3)Based on the transcriptome and RT-qPCR results,rbl2 gene of zebrafish was picked to knockdown by CRISPR/Cas9 technology.The injection embryos were normally raised to sexual maturity.The total DNA was abstracted from the tail of zebrafish.The sequencing results showed that rbl2 gene was knockdown successfully,which laid a foundation for the follow-up experiment.